Vaccines Based on Targeting Antigen to DCIR Expressed on Antigen-Presenting Cells

ABSTRACT

The present invention includes compositions and methods for increasing the effectiveness of antigen presentation using a DCIR-specific antibody or fragment thereof to which an antigen is attached that forms an antibody-antigen complex, wherein the antigen is processed and presented by a dendritic cell that has been contacted with the antibody-antigen complex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/888,032, filed Feb. 2, 2007, the contents of which isincorporated by reference herein in its entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract No.1U19AI057234-0100003 awarded by the NIH. The government has certainrights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of vaccination,and more particularly, to vaccines based on targeting antigen to DCIRexpressed on antigen-presenting cells.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with antigen presentation.

Human vaccines based on dendritic cell (DC)-targeting are a new conceptthat rests on compelling studies in mouse models. Here, small doses ofrelatively weak antigens carried to DC by antibodies directed to certainDC receptors can elicit potent and broad immune responses. To developsuch vaccines for humans needs a better understanding of exactly whichDC receptor should be used for this antigen-targeting application. Thisis because there is not always exact correspondence between the muse andhuman immune systems, and also because not all potential DC receptorshave been examined carefully for this vaccine application.

Thus, studies that in vitro test various anti-human DC receptor targetshave been initiated, for example, DCs targeted with melanoma antigenpmel17 fused to a human mAb against mannose receptor activated T cellsin the context of HLA class I and class II molecules (Ramakrishna, Tremlet al. 2004). Also, targeting the model antigen KLH to DCs via ahumanized anti-DC-SIGN mAb effectively induced antigen-specific naive aswell as dose-sparing recall T cell responses (Tacken, de Vries et al.2005). Besides mannose receptor and DC-SIGN, human DCs express otherreceptors known to be involved in antigen capture. Many of these areC-type lectin receptors (CLRs) including LOX-1, DEC205, DC-ASGPR,Langerin, DCIR, BDCA-2, DECTIN-1, and CLEC-6. These CLRs are differentlyexpressed by distinct subsets of DC and their expression can vary withthe state of DC maturation (Figdor, van Kooyk et al. 2002; Geijtenbeek,van Vliet et al. 2004).

DC subsets stimulate distinct immune responses, and therefore, targetingantigen to these subsets via differentially expressed receptor shouldelicit different immune responses (Shortman and Liu 2002). Furthermore,different receptors on the same DC subset may direct the antigen toseparate processing pathways (Trombetta and Mellman 2005). Lastly, someof these receptors are not intrinsically activating (e.g., DEC205(Bonifaz, Bonnyay et al. 2004)), while others may be activating (e.g.,LOX-1 (Delneste, Magistrelli et al. 2002)) or have not been studiedthoroughly. The importance of DC-activation concomitant with antigenuptake is not known. But if this is beneficial, the DC-activation viathe targeting mAb would simplify formulation of targeting vaccines.

SUMMARY OF THE INVENTION

In the context of these considerations, the present inventors haverecognized an urgent need for a systematic comparison to define the mostappropriate human DC-targeting receptors for desired immune outcomes byexploring in detail in vitro, CD⁴⁺ and CD⁸⁺ T cell naive and recallresponses. This application describes the special and unexpectedcharacteristics of a particular DC receptor—Dendritic Cell InhibitoryReceptor (DCIR)—which show it to be an ideal receptor for the purpose oftargeting antigens to human DCs for preventative and therapeuticvaccination.

The present invention includes compositions and methods for making andusing vaccine that specifically target (deliver) antigens toantigen-presenting cells for the purpose of eliciting potent and broadimmune responses directed against the antigen. The purpose is primarilyto evoke protective or therapeutic immune responses against the agent(pathogen or cancer) from which the antigen was derived.

More particularly, the present invention includes compositions, methodsand methods for designing and making target specific a singlerecombinant antibody (mAb) that carries one or more antigens in acontrolled modular structure, activating proteins, or other antibodies.The modular rAb carrier of the present invention can be used, e.g., totarget (via one primary recombinant antibody against an internalizinghuman dendritic cell receptor) multiple antigens and/or antigens and anactivating cytokine to dendritic cells (DC). Also, the invention alsoprovides a way of joining two different recombinant mAbs end-to-end in acontrolled and defined manner.

The present invention includes compositions and methods for increasingthe effectiveness of antigen presentation by a DCIR-expressing antigenpresenting cell by isolating and purifying a DCIR-specific antibody orfragment thereof to which a targeted agent is attached that forms anantibody-antigen complex, wherein the agent is processed and presentedby, e.g., a dendritic cell, that has been contacted with theantibody-agent complex. In one embodiment, the antigen presenting cellis a dendritic cell and the DCIR-specific antibody or fragment thereofis bound to one half of a Coherin/Dockerin pair. The DCIR-specificantibody or fragment thereof may also be bound to one half of aCoherin/Dockerin pair and an antigen is bound to the complementary halfof the Coherin/Dockerin pair to form a complex. Non-limiting examplesagents include one or more peptides, proteins, lipids, carbohydrates,nucleic acids and combinations thereof.

The agent may one or more cytokine selected from interleukins,transforming growth factors (TGFs), fibroblast growth factors (FGFs),platelet derived growth factors (PDGFs), epidermal growth factors(EGFs), connective tissue activated peptides (CTAPs), osteogenicfactors, and biologically active analogs, fragments, and derivatives ofsuch growth factors, B/T-cell differentiation factors, B/T-cell growthfactors, mitogenic cytokines, chemotactic cytokines, colony stimulatingfactors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4,IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17,IL18, etc., leptin, myostatin, macrophage stimulating protein,platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL,human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10,PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF,transforming growth factor (TGF) supergene family include the betatransforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bonemorphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB). In another embodiment, the agent comprises an antigen thatis a bacterial, viral, fungal, protozoan or cancer protein.

The present invention also includes compositions and methods forincreasing the effectiveness of antigen presentation by dendritic cellscomprising binding a DCIR-specific antibody or fragment thereof to whichan antigen is attached that forms an antibody-antigen complex, whereinthe antigen is processed and presented by a dendritic cell that has beencontacted with the antibody-antigen complex. Another embodiment is theuse of antibodies or other specific binding molecules directed to DCIRfor delivering antigens to antigen-presenting cells for the purpose ofeliciting protective or therapeutic immune responses. The use ofantigen-targeting reagents specific to DCIR for vaccination via theskin; antigen-targeting reagents specific to DCIR in association withco-administered or linked adjuvant for vaccination or use forantigen-targeting (vaccination) purposes of specific antigens which canbe expressed as recombinant antigen-antibody fusion proteins.

Another embodiment includes a method for increasing the effectiveness ofdendritic cells by isolating patient dendritic cells; exposing thedendritic cells to activating amounts of anti-DCIR antibodies orfragments thereof and antigen to form antigen-loaded, activateddendritic cells; and reintroducing the antigen-loaded, activateddendritic cells into the patient. The antigen may be a bacterial, viral,fungal, protozoan or cancer protein. The present invention also includesan anti-DCIR immunoglobulin or portion thereof that is secreted frommammalian cells and an antigen bound to the immunoglobulin. Theimmunoglobulin is bound to one half of a cohesin/dockerin domain, or itmay also include a complementary half of the cohesin-dockerin bindingpair bound to an antigen that forms a complex with the modular rAbcarrier, or a complementary half of the cohesin-dockerin binding pairthat is a fusion protein with an antigen. The antigen specific domainmay be a full length antibody, an antibody variable region domain, anFab fragment, a Fab′ fragment, an F(ab)2 fragment, and Fv fragment, andFabc fragment and/or a Fab fragment with portions of the Fc domain. Theanti-DCIR immunoglobulin may also be bound to a toxin selected fromwherein the toxin is selected from the group consisting of a radioactiveisotope, metal, enzyme, botulin, tetanus, ricin, cholera, diphtheria,aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcalenterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin,alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom. Theantigen may be a fusion protein with the immunoglobulin or boundchemically covalently or not.

Another embodiment is a vaccine with a DCIR-specific antibody orfragment thereof to which an antigen is attached that forms anantibody-antigen complex, wherein the antigen is processed and presentedby a dendritic cell that has been contacted with the antibody-antigencomplex.

The novel antibodies of the present invention were also able to shownovel tissue distribution information. Due to their specific affinity,it was found the the anti-DCIR antibodies of the present inventionbinding monkey DCIR, and are effective for using anti-DCIR-Flu mltargeting for expanding Flu ml-specific CD8 cells in vivo [hu-mouse],and in vitro targeting of ex vivo human skin cells. Furthermore, it wasfound that a complex of carbohydrate ligands for DCIR can be used assurrogates for anti-DCIR agents for antigen delivery. Therefore, anotherembodiment of the present invention is a T cell antigen that includes anantigenic T cell epitope peptide bound to at least a portion of a glycanthat includesNeu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12that binds specifically to DCIR. The glycan (and derivatives thereof)can also be used alone or in combination to block DCIR binding.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 shows the high affinity interaction of the mAbs with DCIR boundto plate by Capture;

FIG. 2 shows the high affinity interaction of the mAbs with DCIR boundto plate by Direct ELISA;

FIG. 3 shows the binding of the high affinity antibodies for use inFACS;

FIG. 4 shows that DCIR is also expressed on three human DC subtypesisolated directly from human skin;

FIG. 5 shows DCIR-specific staining of a population of cells surroundinga germinal center within a human tonsil;

FIG. 6 shows an example of the cross-Flu M1 protein and the mAb to DCIR;

FIG. 7 shows the cross-linking of Coh.Flu M1 to Anti-DCIR_(—)2C9 mAb;

FIG. 8 shows that Flu M1 cross-linked to anti-DCIR mAb induces theexpansion of Flu M1-specific CD8+ T cells more efficiently than Flu M1protein unlinked to mAb;

FIG. 9 shows that Flu M1 cross-linked to anti-DCIR mAb induces theexpansion of Flu M1-specific CD8+ T cells more efficiently via LCs thanInt-DCs;

FIG. 10 shows such H+L chain vectors encoding chimeric mouse-human rAbscorresponding to a number of different anti-DCIR mAbs co-transfectedinto 293 cells and assayed by anti-human FC ELISA for secretion of rAbinto the culture supernatant;

FIG. 11 shows that Coh.Flu M1 linked to anti-DCIR.Doc rAb bindsspecifically to GM/IL-15 human DC;

FIG. 12 shows that the Coh.Flu M1 linked to anti-DC-SIGN/L.Doc oranti-DCIR.Doc rAb binds and is internalized into to GM-CSF/IL-4 humanDC;

FIG. 13 shows that the Anti-DCIR.Doc:Coh.Flu complex is more efficientat expanding Flu M1-specific CD8+ T cells than other [anti-DC receptorrAbs.Doc:Coh.Flu M1] complexes;

FIG. 14 shows that the Anti-DCIR.Doc:Coh.Flu complex administered for 1day is more efficient at expanding Flu M1-specific CD8+ T cells thanother [anti-DC receptor rAbs.Doc:Coh.Flu M1] complexes;

FIG. 15 shows that various antigens expressed as fusions to theC-terminus of rAb H chain have intrinsic effects on the secretion ofrAb.antigen;

FIG. 16 shows the Anti-DCIR.Flu HA5 rAbs are secreted at variousefficiencies depending on the nature of the variable regions;

FIG. 17 shows that Anti-DCIR mAb enhances priming of HIVantigen-specific CD8+ cells;

FIG. 18 shows the Anti-DCIR mAb enhances priming of HIV antigen-specificCD8+ cells;

FIG. 19 shows immunohistochemistry analysis of DCIR distribution inhuman epithelial sheet;

FIGS. 20A-20D shows monoclonal antibodies to DCIR Antigen, specifically,affinity to DCIR;

FIG. 21 shows the cross-reactivity of anti-DCIR mAbs to Rhesus macaqueDCIR;

FIG. 22 is a graph that shows the binding of DCIR ectodomain to specificglycan structures;

FIG. 23A to 23C show that DCIR is a global target for all blood DCsubsets; and

FIG. 24 shows that demonstrate that vaccination with DCIR-FluM1 permitsgeneration of FluM1 specific recall CD8+ T cell immunity.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

Dendritic cells (DCs) are antigen-presenting cells that play a key rolein regulating antigen-specific immunity (Mellman and Steinman 2001),(Banchereau, Briere et al. 2000), (Cella, Sallusto et al. 1997). DCscapture antigens, process them into peptides, and present these to Tcells. Therefore delivering antigens directly to DC is a focus area forimproving vaccines. One such example is the development of DC-basedvaccines using ex-vivo antigen-loading of autologous DCs that are thenre-administrated to patients (Banchereau, Schuler-Thurner et al. 2001),(Steinman and Dhodapkar 2001). Another strategy to improve vaccineefficacy is specific targeting to DC of antigen conjugated to antibodiesagainst internalizing DC-specific receptors. The potential of targetingDCfor vaccination is highlighted by key mouse studies. In vivo,targeting with an anti-LOX-1 mAb coupled to ovalbumin (OVA) induced aprotective CD8+ T cell response, via exogenous antigencross-presentation toward the MHC class I pathway (Delneste, Magistrelliet al. 2002). Also, OVA conjugated to anti-DEC205 mAb in combinationwith a CD40L maturation stimulus enhanced the MHC class I-restrictedpresentation by DCs in vivo and led to the durable formation of effectormemory CD8+ T cells (Bonifaz, Bonnyay et al. 2004). Both these studiesshowed dramatic dose-sparing (i.e., strong immune-responses at very lowantigen doses) and suggested broader responses than normally seen withother types of OVA immunization. Recent work with targeting of HIV gagantigen to DC via DEC205 has extended these concepts to a clinicallyrelevant antigen and confirmed the tenents of targeting antigen toDC—dramatic dose-sparing, protective responses from a singlevaccination, and expansion of antigen-specific T cells in both the CD8and CD4 compartments (Trumpfheller, Finke et al. 2006).

The present invention provides for the complexing of multiple antigensor proteins (engineered, expressed, and purified independently from theprimary mAb) in a controlled, multivariable fashion, to one singleprimary recombinant mAb. Presently, there are methods for engineeringsite-specific biotinylation sites that provide for the addition ofdifferent proteins (each engineered separately linked to streptavidin)to the one primary mAb. However, the present invention provides foraddition to the primary mAb of multiple combinations, in fixed equimolarratios and locations, of separately engineered proteins.

As used herein, the term “modular rAb carrier” is used to describe arecombinant antibody system that has been engineered to provide thecontrolled modular addition of diverse antigens, activating proteins, orother antibodies to a single recombinant monoclonal antibody (mAb). TherAb may be a monoclonal antibody made using standard hybridomatechniques, recombinant antibody display, humanized monoclonalantibodies and the like. The modular rAb carrier can be used to, e.g.,target (via one primary recombinant antibody against an internalizingreceptor, e.g., a human dendritic cell receptor) multiple antigensand/or antigens and an activating cytokine to dendritic cells (DC). Themodular rAb carrier may also be used to join two different recombinantmAbs end-to-end in a controlled and defined manner.

The antigen binding portion of the “modular rAb carrier” may be one ormore variable domains, one or more variable and the first constantdomain, an Fab fragment, a Fab′ fragment, an F(ab)₂ fragment, and Fvfragment, and Fabc fragment and/or a Fab fragment with portions of theFc domain to which the cognate modular binding portions are added to theamino acid sequence and/or bound. The antibody for use in the modularrAb carrier can be of any isotype or class, subclass or from any source(animal and/or recombinant).

In one non-limiting example, the modular rAb carrier is engineered tohave one or more modular cohesin-dockerin protein domains for makingspecific and defined protein complexes in the context of engineeredrecombinant mAbs. The mAb is a portion of a fusion protein that includesone or more modular cohesin-dockerin protein domains carboxy from theantigen binding domains of the mAb. The cohesin-dockerin protein domainsmay even be attached post-translationally, e.g., by using chemicalcross-linkers and/or disulfide bonding.

The term “antigen” as used herein refers to a molecule that can initiatea humoral and/or cellular immune response in a recipient of the antigen.Antigen may be used in two different contexts with the presentinvention: as a target for the antibody or other antigen recognitiondomain of the rAb or as the molecule that is carried to and/or into acell or target by the rAb as part of a dockerin/cohesin-moleculecomplement to the modular rAb carrier. The antigen is usually an agentthat causes a disease for which a vaccination would be advantageoustreatment. When the antigen is presented on MHC, the peptide is oftenabout 8 to about 25 amino acids. Antigens include any type of biologicmolecule, including, for example, simple intermediary metabolites,sugars, lipids and hormones as well as macromolecules such as complexcarbohydrates, phospholipids, nucleic acids and proteins. Commoncategories of antigens include, but are not limited to, viral antigens,bacterial antigens, fungal antigens, protozoal and other parasiticantigens, tumor antigens, antigens involved in autoimmune disease,allergy and graft rejection, and other miscellaneous antigens.

The modular rAb carrier is able to carry any number of active agents,e.g., antibiotics, anti-infective agents, antiviral agents, anti-tumoralagents, antipyretics, analgesics, anti-inflammatory agents, therapeuticagents for osteoporosis, enzymes, cytokines, anticoagulants,polysaccharides, collagen, cells, and combinations of two or more of theforegoing active agents. Examples of antibiotics for delivery using thepresent invention include, without limitation, tetracycline,aminoglycosides, penicillins, cephalosporins, sulfonamide drugs,chloramphenicol sodium succinate, erythromycin, vancomycin, lincomycin,clindamycin, nystatin, amphotericin B, amantidine, idoxuridine, p-aminosalicyclic acid, isoniazid, rifampin, antinomycin D, mithramycin,daunomycin, adriamycin, bleomycin, vinblastine, vincristine,procarbazine, imidazole carboxamide, and the like.

Examples of anti-tumor agents for delivery using the present inventioninclude, without limitation, doxorubicin, Daunorubicin, taxol,methotrexate, and the like. Examples of antipyretics and analgesicsinclude aspirin, Motrin®, Ibuprofen®, naprosyn, acetaminophen, and thelike.

Examples of anti-inflammatory agents for delivery using the presentinvention include, without limitation, include NSAIDS, aspirin,steroids, dexamethasone, hydrocortisone, prednisolone, Diclofenac Na,and the like.

Examples of therapeutic agents for treating osteoporosis and otherfactors acting on bone and skeleton include for delivery using thepresent invention include, without limitation, calcium, alendronate,bone GLa peptide, parathyroid hormone and its active fragments, histoneH4-related bone formation and proliferation peptide and mutations,derivatives and analogs thereof.

Examples of enzymes and enzyme cofactors for delivery using the presentinvention include, without limitation, pancrease, L-asparaginase,hyaluronidase, chymotrypsin, trypsin, tPA, streptokinase, urokinase,pancreatin, collagenase, trypsinogen, chymotrypsinogen, plasminogen,streptokinase, adenyl cyclase, superoxide dismutase (SOD), and the like.

Examples of cytokines for delivery using the present invention include,without limitation, interleukins, transforming growth factors (TGFs),fibroblast growth factors (FGFs), platelet derived growth factors(PDGFs), epidermal growth factors (EGFs), connective tissue activatedpeptides (CTAPs), osteogenic factors, and biologically active analogs,fragments, and derivatives of such growth factors. Cytokines may beB/T-cell differentiation factors, B/T-cell growth factors, mitogeniccytokines, chemotactic cytokines, colony stimulating factors,angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18,etc., leptin, myostatin, macrophage stimulating protein,platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL,human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10,PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF or anyfragments or combinations thereof. Other cytokines include members ofthe transforming growth factor (TGF) supergene family include the betatransforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bonemorphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (forexample, fibroblast growth factor (FGF), epidermal growth factor (EGF),platelet-derived growth factor (PDGF), insulin-like growth factor(IGF)); Inhibins (for example, Inhibin A, Inhibin B); growthdifferentiating factors (for example, GDF-1); and Activins (for example,Activin A, Activin B, Activin AB).

Examples of growth factors for delivery using the present inventioninclude, without limitation, growth factors that can be isolated fromnative or natural sources, such as from mammalian cells, or can beprepared synthetically, such as by recombinant DNA techniques or byvarious chemical processes. In addition, analogs, fragments, orderivatives of these factors can be used, provided that they exhibit atleast some of the biological activity of the native molecule. Forexample, analogs can be prepared by expression of genes altered bysite-specific mutagenesis or other genetic engineering techniques.

Examples of anticoagulants for delivery using the present inventioninclude, without limitation, include warfarin, heparin, Hirudin, and thelike. Examples of factors acting on the immune system include fordelivery using the present invention include, without limitation,factors which control inflammation and malignant neoplasms and factorswhich attack infective microorganisms, such as chemotactic peptides andbradykinins.

Examples of viral antigens include, but are not limited to, e.g.,retroviral antigens such as retroviral antigens from the humanimmunodeficiency virus (HIV) antigens such as gene products of the gag,pol, and env genes, the Nef protein, reverse transcriptase, and otherHIV components; hepatitis viral antigens such as the S, M, and Lproteins of hepatitis B virus, the pre-S antigen of hepatitis B virus,and other hepatitis, e.g., hepatitis A, B, and C, viral components suchas hepatitis C viral RNA; influenza viral antigens such as hemagglutininand neuraminidase and other influenza viral components; measles viralantigens such as the measles virus fusion protein and other measlesvirus components; rubella viral antigens such as proteins E1 and E2 andother rubella virus components; rotaviral antigens such as VP7sc andother rotaviral components; cytomegaloviral antigens such as envelopeglycoprotein B and other cytomegaloviral antigen components; respiratorysyncytial viral antigens such as the RSV fusion protein, the M2 proteinand other respiratory syncytial viral antigen components; herpes simplexviral antigens such as immediate early proteins, glycoprotein D, andother herpes simplex viral antigen components; varicella zoster viralantigens such as gpI, gpII, and other varicella zoster viral antigencomponents; Japanese encephalitis viral antigens such as proteins E,M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitisviral antigen components; rabies viral antigens such as rabiesglycoprotein, rabies nucleoprotein and other rabies viral antigencomponents. See Fundamental Virology, Second Edition, eds. Fields, B. N.and Knipe, D. M. (Raven Press, New York, 1991) for additional examplesof viral antigens.

Antigenic targets that may be delivered using the rAb-DC/DC-antigenvaccines of the present invention include genes encoding antigens suchas viral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picornavirus, coronavirus, togavirus,flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenavirus, reovirus, retrovirus, papilomavirus, parvovirus,herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Other viraltargets include influenza, herpes simplex virus 1 and 2, measles,dengue, smallpox, polio or HIV. Pathogens include trypanosomes,tapeworms, roundworms, helminthes, malaria. Tumor markers, such as fetalantigen or prostate specific antigen, may be targeted in this manner.Other examples include: HIV env proteins and hepatitis B surfaceantigen. Administration of a vector according to the present inventionfor vaccination purposes would require that the vector-associatedantigens be sufficiently non-immunogenic to enable long term expressionof the transgene, for which a strong immune response would be desired.In some cases, vaccination of an individual may only be requiredinfrequently, such as yearly or biennially, and provide long termimmunologic protection against the infectious agent. Specific examplesof organisms, allergens and nucleic and amino sequences for use invectors and ultimately as antigens with the present invention may befound in U.S. Pat. No. 6,541,011, relevant portions incorporated hereinby reference, in particular, the tables that match organisms andspecific sequences that may be used with the present invention.

Bacterial antigens for use with the rAb vaccine disclosed hereininclude, but are not limited to, e.g., bacterial antigens such aspertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3,adenylate cyclase and other pertussis bacterial antigen components;diptheria bacterial antigens such as diptheria toxin or toxoid and otherdiptheria bacterial antigen components; tetanus bacterial antigens suchas tetanus toxin or toxoid and other tetanus bacterial antigencomponents; streptococcal bacterial antigens such as M proteins andother streptococcal bacterial antigen components; gram-negative bacillibacterial antigens such as lipopolysaccharides and other gram-negativebacterial antigen components, Mycobacterium tuberculosis bacterialantigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDamajor secreted protein, antigen 85A and other mycobacterial antigencomponents; Helicobacter pylori bacterial antigen components;pneumococcal bacterial antigens such as pneumolysin, pneumococcalcapsular polysaccharides and other pneumococcal bacterial antigencomponents; haemophilus influenza bacterial antigens such as capsularpolysaccharides and other haemophilus influenza bacterial antigencomponents; anthrax bacterial antigens such as anthrax protectiveantigen and other anthrax bacterial antigen components; rickettsiaebacterial antigens such as rompA and other rickettsiae bacterial antigencomponent. Also included with the bacterial antigens described hereinare any other bacterial, mycobacterial, mycoplasmal, rickettsial, orchlamydial antigens. Partial or whole pathogens may also be: haemophilusinfluenza; Plasmodium falciparum; neisseria meningitidis; streptococcuspneumoniae; neisseria gonorrhoeae; salmonella serotype typhi; shigella;vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis;lyme disease; Yersinia pestis; west nile virus; yellow fever; tularemia;hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1and HPIV 3; adenovirus; small pox; allergies and cancers.

Fungal antigens for use with compositions and methods of the inventioninclude, but are not limited to, e.g., candida fungal antigencomponents; histoplasma fungal antigens such as heat shock protein 60(HSP60) and other histoplasma fungal antigen components; cryptococcalfungal antigens such as capsular polysaccharides and other cryptococcalfungal antigen components; coccidiodes fungal antigens such as spheruleantigens and other coccidiodes fungal antigen components; and tineafungal antigens such as trichophytin and other coccidiodes fungalantigen components.

Examples of protozoal and other parasitic antigens include, but are notlimited to, e.g., plasmodium falciparum antigens such as merozoitesurface antigens, sporozoite surface antigens, circumsporozoiteantigens, gametocyte/gamete surface antigens, blood-stage antigen pf155/RESA and other plasmodial antigen components; toxoplasma antigenssuch as SAG-1, p30 and other toxoplasmal antigen components;schistosomae antigens such as glutathione-S-transferase, paramyosin, andother schistosomal antigen components; leishmania major and otherleishmaniae antigens such as gp63, lipophosphoglycan and its associatedprotein and other leishmanial antigen components; and trypanosoma cruziantigens such as the 75-77 kDa antigen, the 56 kDa antigen and othertrypanosomal antigen components.

Antigen that can be targeted using the rAb of the present invention willgenerally be selected based on a number of factors, including:likelihood of internalization, level of immune cell specificity, type ofimmune cell targeted, level of immune cell maturity and/or activationand the like. Examples of cell surface markers for dendritic cellsinclude, but are not limited to, MHC class I, MHC Class II, B7-2, CD18,CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56,DCIR and/or ASPGR and the like; while in some cases also having theabsence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56,and/or CD57. Examples of cell surface markers for antigen presentingcells include, but are not limited to, MHC class I, MHC Class II, CD40,CD45, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1 and/or Fcγreceptor. Examples of cell surface markers for T cells include, but arenot limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 andHLA-DR.

Target antigens on cell surfaces for delivery includes thosecharacteristic of tumor antigens typically will be derived from the cellsurface, cytoplasm, nucleus, organelles and the like of cells of tumortissue. Examples of tumor targets for the antibody portion of thepresent invention include, without limitation, hematological cancerssuch as leukemias and lymphomas, neurological tumors such asastrocytomas or glioblastomas, melanoma, breast cancer, lung cancer,head and neck cancer, gastrointestinal tumors such as gastric or coloncancer, liver cancer, pancreatic cancer, genitourinary tumors suchcervix, uterus, ovarian cancer, vaginal cancer, testicular cancer,prostate cancer or penile cancer, bone tumors, vascular tumors, orcancers of the lip, nasopharynx, pharynx and oral cavity, esophagus,rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder,kidney, brain and other parts of the nervous system, thyroid, Hodgkin'sdisease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.

Examples of antigens that may be delivered alone or in combination toimmune cells for antigen presentation using the present inventioninclude tumor proteins, e.g., mutated oncogenes; viral proteinsassociated with tumors; and tumor mucins and glycolipids. The antigensmay be viral proteins associated with tumors would be those from theclasses of viruses noted above. Certain antigens may be characteristicof tumors (one subset being proteins not usually expressed by a tumorprecursor cell), or may be a protein which is normally expressed in atumor precursor cell, but having a mutation characteristic of a tumor.Other antigens include mutant variant(s) of the normal protein having analtered activity or subcellular distribution, e.g., mutations of genesgiving rise to tumor antigens.

Specific non-limiting examples of tumor antigens include: CEA, prostatespecific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC(Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc,tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron Vsequence (N-acetylglucoaminyltransferase V intron V sequence), ProstateCa psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virusnuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53,lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, theimmunogenic molecule can be an autoantigen involved in the initiationand/or propagation of an autoimmune disease, the pathology of which islargely due to the activity of antibodies specific for a moleculeexpressed by the relevant target organ, tissue, or cells, e.g., SLE orMG. In such diseases, it can be desirable to direct an ongoingantibody-mediated (i.e., a Th2-type) immune response to the relevantautoantigen towards a cellular (i.e., a Th1-type) immune response.Alternatively, it can be desirable to prevent onset of or decrease thelevel of a Th2 response to the autoantigen in a subject not having, butwho is suspected of being susceptible to, the relevant autoimmunedisease by prophylactically inducing a Th1 response to the appropriateautoantigen. Autoantigens of interest include, without limitation: (a)with respect to SLE, the Smith protein, RNP ribonucleoprotein, and theSS-A and SS-B proteins; and (b) with respect to MG, the acetylcholinereceptor. Examples of other miscellaneous antigens involved in one ormore types of autoimmune response include, e.g., endogenous hormonessuch as luteinizing hormone, follicular stimulating hormone,testosterone, growth hormone, prolactin, and other hormones.

Antigens involved in autoimmune diseases, allergy, and graft rejectioncan be used in the compositions and methods of the invention. Forexample, an antigen involved in any one or more of the followingautoimmune diseases or disorders can be used in the present invention:diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis),multiple sclerosis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjogren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.Examples of antigens involved in autoimmune disease include glutamicacid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelinproteolipid protein, acetylcholine receptor components, thyroglobulin,and the thyroid stimulating hormone (TSH) receptor. Examples of antigensinvolved in allergy include pollen antigens such as Japanese cedarpollen antigens, ragweed pollen antigens, rye grass pollen antigens,animal derived antigens such as dust mite antigens and feline antigens,histocompatiblity antigens, and penicillin and other therapeutic drugs.Examples of antigens involved in graft rejection include antigeniccomponents of the graft to be transplanted into the graft recipient suchas heart, lung, liver, pancreas, kidney, and neural graft components.The antigen may be an altered peptide ligand useful in treating anautoimmune disease.

As used herein, the term “epitope(s)” refer to a peptide or proteinantigen that includes a primary, secondary or tertiary structure similarto an epitope located within any of a number of pathogen polypeptidesencoded by the pathogen DNA or RNA. The level of similarity willgenerally be to such a degree that monoclonal or polyclonal antibodiesdirected against such polypeptides will also bind to, react with, orotherwise recognize, the peptide or protein antigen. Various immunoassaymethods may be employed in conjunction with such antibodies, such as,for example, Western blotting, ELISA, RIA, and the like, all of whichare known to those of skill in the art. The identification of pathogenepitopes, and/or their functional equivalents, suitable for use invaccines is part of the present invention. Once isolated and identified,one may readily obtain functional equivalents. For example, one mayemploy the methods of Hopp, as taught in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. The methods described in several other papers, andsoftware programs based thereon, can also be used to identify epitopiccore sequences (see, for example, Jameson and Wolf, 1988; Wolf et al.,1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these“epitopic core sequences” may then be readily incorporated intopeptides, either through the application of peptide synthesis orrecombinant technology.

The preparation of vaccine compositions that includes the nucleic acidsthat encode antigens of the invention as the active ingredient, may beprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinfection can also be prepared. The preparation may be emulsified,encapsulated in liposomes. The active immunogenic ingredients are oftenmixed with carriers which are pharmaceutically acceptable and compatiblewith the active ingredient.

The term “pharmaceutically acceptable carrier” refers to a carrier thatdoes not cause an allergic reaction or other untoward effect in subjectsto whom it is administered. Suitable pharmaceutically acceptablecarriers include, for example, one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the vaccine can containminor amounts of auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, and/or adjuvants which enhance theeffectiveness of the vaccine. Examples of adjuvants that may beeffective include but are not limited to: aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, whichcontains three components extracted from bacteria, monophosporyl lipidA, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%squalene/Tween 80 emulsion. Other examples of adjuvants include DDA(dimethyldioctadecylammonium bromide), Freund's complete and incompleteadjuvants and QuilA. In addition, immune modulating substances such aslymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducerssuch as poly I:C can be used in combination with adjuvants describedherein.

Pharmaceutical products that may include a naked polynucleotide with asingle or multiple copies of the specific nucleotide sequences that bindto specific DNA-binding sites of the apolipoproteins present on plasmalipoproteins as described in the current invention. The polynucleotidemay encode a biologically active peptide, antisense RNA, or ribozyme andwill be provided in a physiologically acceptable administrable form.Another pharmaceutical product that may spring from the currentinvention may include a highly purified plasma lipoprotein fraction,isolated according to the methodology, described herein from either thepatients blood or other source, and a polynucleotide containing singleor multiple copies of the specific nucleotide sequences that bind tospecific DNA-binding sites of the apolipoproteins present on plasmalipoproteins, prebound to the purified lipoprotein fraction in aphysiologically acceptable, administrable form.

Yet another pharmaceutical product may include a highly purified plasmalipoprotein fraction which contains recombinant apolipoprotein fragmentscontaining single or multiple copies of specific DNA-binding motifs,prebound to a polynucleotide containing single or multiple copies of thespecific nucleotide sequences, in a physiologically acceptableadministrable form. Yet another pharmaceutical product may include ahighly purified plasma lipoprotein fraction which contains recombinantapolipoprotein fragments containing single or multiple copies ofspecific DNA-binding motifs, prebound to a polynucleotide containingsingle or multiple copies of the specific nucleotide sequences, in aphysiologically acceptable administrable form.

The dosage to be administered depends to a great extent on the bodyweight and physical condition of the subject being treated as well asthe route of administration and frequency of treatment. A pharmaceuticalcomposition that includes the naked polynucleotide prebound to a highlypurified lipoprotein fraction may be administered in amounts rangingfrom 1 μg to 1 mg polynucleotide and 1 μg to 100 mg protein.

Administration of an rAb and rAb complexes a patient will follow generalprotocols for the administration of chemotherapeutics, taking intoaccount the toxicity, if any, of the vector. It is anticipated that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, as well as surgical intervention, maybe applied in combination with the described gene therapy.

Where clinical application of a gene therapy is contemplated, it will benecessary to prepare the complex as a pharmaceutical compositionappropriate for the intended application. Generally this will entailpreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals. One also will generally desire to employ appropriatesalts and buffers to render the complex stable and allow for complexuptake by target cells.

Aqueous compositions of the present invention may include an effectiveamount of the compound, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. Such compositions can also bereferred to as inocula. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions. The compositions of the present invention mayinclude classic pharmaceutical preparations. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Disease States. Depending on the particular disease to be treated,administration of therapeutic compositions according to the presentinvention will be via any common route so long as the target tissue isavailable via that route in order to maximize the delivery of antigen toa site for maximum (or in some cases minimum) immune response.Administration will generally be by orthotopic, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Other areas for delivery include: oral, nasal, buccal, rectal, vaginalor topical. Topical administration would be particularly advantageousfor treatment of skin cancers. Such compositions would normally beadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

Vaccine or treatment compositions of the invention may be administeredparenterally, by injection, for example, either subcutaneously orintramuscularly. Additional formulations which are suitable for othermodes of administration include suppositories, and in some cases, oralformulations or formulations suitable for distribution as aerosols. Inthe case of the oral formulations, the manipulation of T-cell subsetsemploying adjuvants, antigen packaging, or the addition of individualcytokines to various formulation that result in improved oral vaccineswith optimized immune responses. For suppositories, traditional bindersand carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of active ingredient, preferably 25-70%.

The antigen encoding nucleic acids of the invention may be formulatedinto the vaccine or treatment compositions as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or with organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine or treatment compositions are administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. The quantity to beadministered depends on the subject to be treated, including, e.g.,capacity of the subject's immune system to synthesize antibodies, andthe degree of protection or treatment desired. Suitable dosage rangesare of the order of several hundred micrograms active ingredient pervaccination with a range from about 0.1 mg to 1000 mg, such as in therange from about 1 mg to 300 mg, and preferably in the range from about10 mg to 50 mg. Suitable regiments for initial administration andbooster shots are also variable but are typified by an initialadministration followed by subsequent inoculations or otheradministrations. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and may bepeculiar to each subject. It will be apparent to those of skill in theart that the therapeutically effective amount of nucleic acid moleculeor fusion polypeptides of this invention will depend, inter alia, uponthe administration schedule, the unit dose of antigen administered,whether the nucleic acid molecule or fusion polypeptide is administeredin combination with other therapeutic agents, the immune status andhealth of the recipient, and the therapeutic activity of the particularnucleic acid molecule or fusion polypeptide.

The compositions can be given in a single dose schedule or in a multipledose schedule. A multiple dose schedule is one in which a primary courseof vaccination may include, e.g., 1-10 separate doses, followed by otherdoses given at subsequent time intervals required to maintain and orreinforce the immune response, for example, at 1-4 months for a seconddose, and if needed, a subsequent dose(s) after several months. Periodicboosters at intervals of 1-5 years, usually 3 years, are desirable tomaintain the desired levels of protective immunity. The course of theimmunization can be followed by in vitro proliferation assays ofperipheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST-CF, andby measuring the levels of IFN-γ released from the primed lymphocytes.The assays may be performed using conventional labels, such asradionucleotides, enzymes, fluorescent labels and the like. Thesetechniques are known to one skilled in the art and can be found in U.S.Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant portionsincorporated by reference.

The modular rAb carrier and/or conjugated rAb carrier-(cohesion/dockerinand/or dockerin-cohesin)-antigen complex (rAb-DC/DC-antigen vaccine) maybe provided in one or more “unit doses” depending on whether the nucleicacid vectors are used, the final purified proteins, or the final vaccineform is used. Unit dose is defined as containing apredetermined-quantity of the therapeutic composition calculated toproduce the desired responses in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, and the particular route and formulation, are within theskill of those in the clinical arts. The subject to be treated may alsobe evaluated, in particular, the state of the subject's immune systemand the protection desired. A unit dose need not be administered as asingle injection but may include continuous infusion over a set periodof time. Unit dose of the present invention may conveniently may bedescribed in terms of DNA/kg (or protein/Kg) body weight, with rangesbetween about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000or more mg/DNA or protein/kg body weight are administered. Likewise theamount of rAb-DC/DC-antigen vaccine delivered can vary from about 0.2 toabout 8.0 mg/kg body weight. Thus, in particular embodiments, 0.4 mg,0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg,5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg of the vaccine may bedelivered to an individual in vivo. The dosage of rAb-DC/DC-antigenvaccine to be administered depends to a great extent on the weight andphysical condition of the subject being treated as well as the route ofadministration and the frequency of treatment. A pharmaceuticalcomposition that includes a naked polynucleotide prebound to a liposomalor viral delivery vector may be administered in amounts ranging from 1μg to 1 mg polynucleotide to 1 μg to 100 mg protein. Thus, particularcompositions may include between about 1 μg, 5 μg, 10 μg, 20 μg, 30 μg,40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500μg, 600 μg, 700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or proteinthat is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0 μg, 40 μg50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg vector.

The present invention was tested in an in vitro cellular system thatmeasures immune stimulation of human Flu-specific T cells by dendriticcells to which Flu antigen has been targeted. The results shown hereindemonstrate the specific expansion of such antigen specific cells atdoses of the antigen which are by themselves ineffective in this system.

The present invention may also be used to make a modular rAb carrierthat is, e.g., a recombinant humanized mAb (directed to a specific humandendritic cell receptor) complexed with protective antigens from Ricin,Anthrax toxin, and Staphylococcus B enterotoxin. The potential marketfor this entity is vaccination of all military personnel and storedvaccine held in reserve to administer to large population centers inresponse to any biothreat related to these agents. The invention hasbroad application to the design of vaccines in general, both for humanand animal use. Industries of interest include the pharmaceutical andbiotechnology industries.

General methods—Restriction and DNA modification enzymes were from NEB.Plasmid and DNA fragment purification was with Qiagen products. SDS-PAGEwas via 4-12% Bis-Tris gels stained with Simply Blue (Invitrogen).Chromatography columns and resins were from GE Healthcare. Plasmidconstructs were confirmed by DNA sequencing (MCLAB). DNA primers werefrom Operon or Midland Certified Reagent Company. Sequence analysis wasvia Sequencher (Gene Codes). Protein concentrations based on calculatedextinction coefficient predicted by the ProtParam tool (2005) weremeasured by UV absorption (NanoDrop ND-1000). The sequences are providedin the Sequence Listing SEQ ID NOS.: 1-39, incorp[orated herein byreference, which are alignments anti-DCIR mAb Heavy (SEQ ID NOS.: 1-17)and Light chain signal peptide and variable region sequences (SEQ IDNOS.:18-39). Predicted N-terminal signal peptide region, sequencedifferences between variants or between closely related sequences weredetermined using Sequencher.

Sequence of the C-terminal extension to the Cohesin domain ofCohesin-Flex-hMART-1-PeptideA-6× His protein. The immunodominant peptidesequence peptide is underlined and bold residues bounding the peptideare native to the antigen sequence. C-terminal His tags are tofacilitate purification via Ni++ affinity chromatography. C186Cohesin-Flex-hMART-1-Peptide A-6× His:

(SEQ ID NO.:40) ASDTTEARHPPVTTPTTDRRKGTTAE ELAGIGILTV ILGGKRTNNSTPTKGEFCRYPSHWRPLEHHHHHH.

Antigen expression constructs—PCR was used to amplify the ORF ofInfluenza A/Puerto Rico/8/34/Mount Sinai (H1N1) M1 protein whileincorporating a Nhe I site distal to the initiator codon and a Not Isite distal to the stop codon. The digested fragment was cloned intopET-28b(+) (Novagen), placing the M1 ORF in-frame with a His6 tag, thusencoding His.Flu M1 protein. The Flu M1 ORF was placed into a similarvector encoding N-terminal protein G precursor B2 domain residues298-352 (gi|124267|) distal to the Nco I site, followed by linkerresidues encoding GGSGGSGGSLD (SEQ ID NO.: 41). This vector expressedProG.Flu M1 protein with a Q246E change. A pET28b (+) derivativeencoding a N-terminal 169 residue cohesin domain from C. thermocelluminserted between the Nco I and Nhe I sites expressed Coh.His. Forexpression of Coh.Flu M1.His, the Flu M1 ORF was inserted between theNhe I and Xho I sites of the above derivative. Coh.PEP.His expressionconstructs were made similarly, except they utilized synthetic DNAsencoding the required sequences. The proteins were expressed in E. colistrain BL21 (DE3) (Novagen) or T7 Express (NEB) grown at 37° C. withselection for kanamycin resistance (40 μg/ml) and shaking at 200rounds/min to mid log phase growth when 120 mg/L IPTG was added. Afterthree hours, the cells were harvested by centrifugation and stored at−80 C. The ProG and Cohesin segments replaced the ectodomain segment inthe AP fusion secretion vector described above, by incorporating a Sal Isite in place of the initiator codon and adding a distal Xho I site forinsertion at the vector Xho I site. An ‘empty’ AP vector was made bydeleting the ectodomain segment. Respectively, these constructs directedsecreted of ProG.AP, Coh.AP and AP.

Expression and purification of recombinant proteins—E. coli cells fromeach 1 L fermentation were resuspended in 30 ml ice-cold 0.1 M NaPO₄ pH7.4 (buffer A, for ProG.Flu M1) or 50 mM Tris, 1 mM EDTA pH 8.0 (bufferB, for all other proteins) with 0.1 ml of protease inhibitor Cocktail II(Calbiochem). The cells were sonicated on ice 2× 5 min at setting 18(Fisher Sonic Dismembrator 60) with a 5 min rest period and then spun at17,000 r.p.m. (Sorvall SA-600) for 20 min at 4° C. For ProG.Flu M1, thesupernatant was passed through 5 ml Q Sepharose equilibrated in buffer Aand then 5 ml hIgG beads were added to the Q flow-through and incubatedwith mixing at 4° C. for 1 h. The bead-bound protein was washed with 50ml cold PBS and eluted with 2×10 ml 0.1 M glycine pH 2.7. The pooledeluates were brought to pH 5 with 0.1 M MES pH 5.0 buffer and run on a 1ml HiTrap S column equilibrated with 50 mM MES pH 5.0 (buffer C). Thecolumn-bound protein was washed extensively with buffer C and elutedwith a 0-1 M NaCl gradient in buffer C. The peak fractions were pooled.For His.Flu M1 purification the 50 ml cell lysate supernatant fractionwas passed through 5 ml Q Sepharose beads and 6.25 ml 160 mM Tris, 40 mMimidazole, 4 M NaCl pH 7.9 was added to the Q Sepharose flow through.This was loaded at 4 ml/min onto a 5 ml HiTrap chelating HP columncharged with Ni⁺⁺. The column-bound protein was washed with 20 mM NaPO₄,300 mM NaCl pH 7.6 (buffer D) followed by another wash with 100 mMH₃COONa pH 4.0. Bound protein was eluted with a gradient from 100 mM to1 M H₃COONa pH 4.0. The peak fractions were pooled and loaded at 4ml/min onto a 5 ml HiTrap S column equilibrated with 100 mM H₃COONa pH4.0, and washed with the equilibration buffer followed by another washwith 50 mM NaPO₄ pH 7.5. Bound protein was eluted with a gradient from0-1 M NaCl in 50 mM NaPO₄ pH 7.5. Peak fractions eluting at about 500 mMNaCl were pooled. Preparations of His.Flu M1 had variable amounts ofnon-full-length products, presumably with C-terminal portions missing.For Coh.Flu M1.His purification, cells from 2 L of culture weresonicated as above, but in buffer B. After centrifugation, 2.5 ml ofTriton X114 was added to the supernatant with incubation on ice for 5min. After further incubation at 25° C. for 5 min, the supernatant wasseparated from the Triton X114 following centrifugation at 25° C. Theextraction was repeated and the supernatant was passed through 5 ml of QSepharose beads and 6.25 ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH7.9 was added to the Q Sepharose flow through. The protein was thenpurified by Ni⁺⁺ chelating chromatography as described above and elutedwith 0-500 mM imidazole in buffer D.

cDNA cloning and expression of chimeric mouse/human mAbs—Total RNA wasprepared from hybridoma cells (RNeasy kit, Qiagen) and used for cDNAsynthesis and PCR (SMART RACE kit, BD Biosciences) using supplied 5′primers and gene specific 3′ primers

mIgGκ, 5′ggatggtgggaagatggatacagttggtgcagcatc3′; (SEQ ID NO.:42) mIgGλ,5′ctaggaacagtcagcacgggacaaactcttctccacagtgtgaccttc3′; (SEQ ID NO.:43)migG1, 5′gtcactggctcagggaaatagcccttgaccaggcatc3′; (SEQ ID NO.:44)mIgG2a, 5′ccaggcatcctagagtcaccgaggagccagt3′; (SEQ ID NO.:45) and mIgG2b,5′ggtgctggaggggacagtcactgagctgctcatagtgt3′. (SEQ ID NO.:46)PCR products were cloned (pCR2.1 TA kit, Invitrogen) and characterizedby DNA sequencing. Using the derived sequences for the mouse H and Lchain V-region cDNAs, specific primers were used to PCR amplify thesignal peptide and V-regions while incorporating flanking restrictionsites for cloning into expression vectors encoding downstream human IgGκor IgG4H regions. The vector for expression of chimeric mVκ-hIgκ wasbuilt by amplifying residues 401-731 (gi|63101937|) flanked by Xho I andNot I sites and inserting this into the Xho I-Not I interval ofpIRES2-DsRed2 (BD Biosciences). PCR was used to amplify the mAb Vkregion from the initiator codon, appending a Nhe I or Spe I site thenCACC, to the region encoding (e.g., residue 126 of gi|76779294|),appending a Xho I site. The PCR fragment was then cloned into the NheI-Not I interval of the above vector. The vector for chimeric mVκ-hIgκusing the mSLAM leader was built by inserting the sequence5′ctagttgctggctaatggaccccaaaggctccctttcctggagaatacttctgtttctctccctggcttttgagttgtcgtacggattaattaagggcccactcgag3′(SEQ ID NO.: 47) into the Nhe I-Xho I interval of the above vector. PCRwas used to amplify the interval between the predicted mature N-terminalcodon (defined using the SignalP 3.0 Server) (Bendtsen, Nielsen et al.2004) and the end of the mVκ region (as defined above) while appending5′tcgtacgga3′. The fragment digested with Bsi WI and Xho I was insertedinto the corresponding sites of the above vector. The control hIgκsequence corresponds to gi|49257887| residues 26-85 and gi|21669402|residues 67-709. The control hIgG4H vector corresponds to residues12-1473 of gi|19684072| with S229P and L236E substitutions, whichstabilize a disulphide bond and abrogate residual FcR binding (Reddy,Kinney et al. 2000), inserted between the pIRES2-DsRed2 vector Bgl IIand Not I sites while adding the sequence 5′gctagctgattaattaa3′ insteadof the stop codon. PCR was used to amplify the mAb VH region from theinitiator codon, appending CACC then a Bgl II site, to the regionencoding residue 473 of gi|19684072|. The PCR fragment was then clonedinto the Bgl II-Apa I interval of the above vector. The vector forchimeric mVH-hIgG4 sequence using the mSLAM leader was built byinserting the sequence5′ctagttgctggctaatggaccccaaaggctccctttcctggagaatacttctgtttctctccctggcttttgagttgtcgtacggattaattaagggccc3′(SEQ ID NO.: 48) into the Nhe I-Apa I interval of the above vector. PCRwas used to amplify the interval between the predicted mature N-terminalcodon and the end of the mVκ region while appending 5′tcgtacgga3′. Thefragment digested with Bsi WI and Apa I was inserted into thecorresponding sites of the above vector.

Various antigen coding sequences flanked by a proximal Nhe I site and adistal Not I site following the stop codon were inserted into the NheI-Pac I-Not I interval of the H chain vectors. Flu HA1-1 was encoded byInfluenza A virus (A/Puerto Rico/8/34(H1N1)) hemagglutinin gi|21693168|residues 82-1025 (with a C982T change) with proximal5′gctagcgatacaacagaacctgcaacacctacaacacctgtaacaa3′ (SEQ ID NO.: 49)sequence (a Nhe I site followed by sequence encoding cipAcohesin-cohesin linker residues) and distal5′caccatcaccatcaccattgagcggccgc3′ (SEQ ID NO.: 50) sequence (encodingHis6, a stop codon, and a Not I site). Flu HA5-1 was encoded bygi|50296052| Influenza A virus (A/Viet Nam/1203/2004(H5N1))hemagglutinin residues 49-990 bound by the same sequences as Flu HA1-1.Doc was encoded by gi|40671| celD residues 1923-2150 with proximal Nhe Iand distal Not I sites. PSA was encoded by gi|34784812| prostatespecific antigen residues 101-832 with proximal sequence5′gctagcgatacaacagaacctgcaacacctacaacacctgtaacaacaccgacaacaacacttctagcgc3′(SEQ ID NO.: 51) (Nhe I site and cipA spacer) and a distal Not I site.Flu M1-PEP was encoded by5′gctagccccattctgagccccctgaccaaaggcattctgggctttgtgtttaccctgaccgtgcccagcgaacgcaagggtatacttggattcgttttcacacttacttaagcggccgc3′(SEQ ID NO.: 52). This and all other peptide-encoding sequences werecreated via mixtures of complimentary synthetic DNA fragments with endscompatible for cloning into Nhe I and Not I-restricted H chain vectors,or Nhe I-Xho I-restricted Coh.His vector. Preferred human codons werealways used, except where restriction sites needed to be incorporated orin CipA spacer sequences.

Production levels of rAb expression constructs were tested in 5 mltransient transfections using 2.5 μg each of the L-chain and H chainconstruct and the protocol described above. Supernatants were analyzedby anti-hIgG ELISA (AffiniPure Goat anti-human IgG (H+L), JacksonImmunoResearch). In tests of this protocol, production of secreted rAbwas independent of H chain and L chain vectors concentration over a2-fold range of each DNA concentration (i.e., the system was DNAsaturated).

Generation of CD34-DCs-CD34+ HPCs were mobilized and collected fromperipheral blood of normal healthy donors, who received subcutaneousrecombinant G-CSF (Neupogen) 10 U/kg/day for 5 days. CD34⁺-HPCs wereobtained with the CEPRATE SC stem cell concentration system (ISOLEX).CD34-DCs were generated by culture at a concentration of 0.5×10⁶/ml inYssel's medium (Irvine Scientific, CA) supplemented with 5% autologousserum, 50 μM 2-β-mercaptoethanol, 1% L-glutamine, 1%penicillin/streptomycin, and the cytokines; GM-CSF (50 ng/ml; ImmunexCorp.), FLT3-L (100 ng/ml; R&D), and TNF-α (10 ng/ml; R&D). Cells weretransferred to fresh medium supplemented with cytokines at day 5 ofculture, and harvested at day 9.

Sorting of CD34-DCs—CD34-derived DCs at day 9 of culture were harvested,and stained with anti-CD1a FITC (Biosource International) and anti-CD14PE (BD Biosciences). CD1a⁺CD14⁻-LCs and CD1a⁻CD14⁺-intDCs were sortedwith FACS Vantage™ (BD Biosciences). Purity was routinely 95-99%.

Purification of autologous CD8⁺ T cells—Autologous CD8+ T cells werepositively selected from PBMCs obtained from the identical donors byusing CD8 magnetic beads (Miltenyi) after depletion with CD14, CD19,CD16, CD56 and CD4 beads. In some experiments, memory CD8+ T cells weresorted as CD8+ CCR7-CD45RA-.

Cross-presentation of Flu M1 protein by CD34-DC subsets to CD8+ Tcells—Bulk or sorted CD34+ DCs subsets, CD1a+ LCs or CD14+ IntDCs (5×10⁴cells/ml) from an HLA-A2 donor, were cultured with purified autologousCD8+ T cells (1×10⁶ cells/ml) in Yssel's medium supplemented with 10%heat-inactivated pooled AB human serum, 10 U/ml IL-7 (R&D) anddecreasing doses of Flu M1 that was cross-linked to an anti-DC antibody.CD40L was added to the culture after 24 h, and IL-2 was added after 3days. Cross presentation efficiency was assessed after 8 or 10 days, byanalyzing the level of antigen-specific CD8+ T cell proliferation, usingspecific Flu M1, HLA-A201/pMI, phycoerythrin-conjugated iTAg MHCTetramer (Beckman Coulter).

Development of anti-human DCIR monoclonal antibodies—Receptorectodomain.hIgG (human IgG1Fc) and HRP (horse radish peroxidase) fusionproteins were produced for immunization of mice and screening of mAbs,respectively. The expression construct for hDCIR ectodomain.IgG wasdescribed previously (Bates, Fournier et al. 1999) and used the mouseSLAM (mSLAM) signal peptide to direct secretion (Bendtsen, Nielsen etal. 2004). The expression vector for hDCIR ectodomain.AP was generatedusing PCR to amplify AP resides 133-1581 (gb|BC009647|) while adding aproximal in-frame Xho I site and a distal TGA stop codon and Not I site.This Xho I-Not I fragment replaced the IgG coding sequence in the abovehDCIR ectodomain.IgG vector. The DCIR.HRP fusion protein vector wasgenerated by cloning gi|208493| residues 14-940 distal to the DCIRectodomain-coding region as defined above.

Expression and purification of recombinant proteins secreted frommammalian cells—Fusion proteins were produced using the FreeStyle™ 293Expression System (Invitrogen) according to the manufacturer's protocol(1 mg total plasmid DNA with 1.3 ml 293 Fectin reagent/L oftransfection). For recombinant antibody (rAb) production, equal amountsof vector encoding the H and L chain were co-transfected. Transfectedcells are cultured for 3 days, the culture supernatant was harvested andfresh media added with continued incubation for two days. The pooledsupernatants were clarified by filtration. Receptor ectodomain.hIgG waspurified by HiTrap protein A affinity chromatography with elution by 0.1M glycine pH 2.7 and then dialyzed versus PBS. rAbs were purifiedsimilarly, by using HiTrap MabSelect™ columns.

Generation of monoclonal antibodies—Mouse mAbs were generated byconventional cell fusion technology. Briefly, 6-week-old BALB/c micewere immunized intraperitoneally with 20 μg of receptorectodomain.hIgGFc fusion protein with Ribi adjuvant, then boosts with 20μg antigen 10 days and 15 days later. After 3 months, the mice wereboosted again three days prior to taking the spleens. Alternately, micewere injected in the footpad with 1-10 μg antigen in Ribi adjuvant every3-4 days over a 30-40 day period. 3-4 days after a final boost, draininglymph nodes were harvested. B cells from spleen or lymph node cells werefused with SP2/O—Ag 14 cells (Shulman, Wilde et al. 1978) usingconventional techniques. ELISA was used to screen hybridoma supernatantsagainst the receptor ectodomain fusion protein compared to the fusionpartner alone, or versus the receptor ectodomain fused to AP (Bates,Fournier et al. 1999). Positive wells were then screened in FACS using293F cells transiently transfected with expression plasmids encodingfull-length receptor cDNAs.

For the development of anti-DCIR mAbs, supernatants from 1000 hybridomaclones screened:

-   -   90 were + on DCIR.Ig vs. Ig ELISA    -   64 were + on DCIR-293 cells by FACS    -   62 FACS+ were ELISA+    -   2 were 293+ (and thus not specific to DCIR)

Biological screen for anti-DCIR mAbs that stimulate cytokine productionby human DC—For DC-targeting purposes, it is potentially desirable tohave the antibody delivering the antigen to the DC and concomitantlyactivating the DC to stimulate a productive immune response against thedelivered antigen. Thus we screened the panel of 62 FACS positiveanti-DCIR hybridoma supernatants directly for DC stimulation activity.CD34+-derived human DC were cultured for 24 hours with the hybridomasupernatants and the DC culture supernatant was assayed 24 hours laterfor the presence of the chemokine MCP-1. The figure below shows thatmany, but not all, hybridoma supernatants elicited specific productionof MCP-1 when compared to controls.

Selected hybridomas (most, but all stimulating MCP-1 production) markedin the figure above with asterisks were single cell cloned and expandedin CELLine flasks (Intergra). Hybridoma supernatants were mixed with anequal volume of 1.5 M glycine, 3 M NaCl, 1× PBS, pH 7.8 and tumbled withMabSelect resin. The resin was washed with binding buffer and elutedwith 0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris, mAbswere dialyzed versus PBS.

Characterization of the pure anti-DCIR mAbs—The pure mAbs were testedfirstly by ELISA (DCIR.Ig bound to the plates, developed withHRP-conjugated anti-human Fc reagents) and by a DCIR.HRP capture assay(mAb bound to the plate, developed with DCIR.HRP fusion protein). FIG. 2shows representative assay results showing high affinity interaction ofthe mAbs with DCIR bound to plate, Capture and Direct ELISA,respectively (controls showing specificity of binding are not shown). Inthe DCIR.HRP capture assay, several (but not all) of the mAbs were ableto capture soluble DCIR.HRP to the plate surface. These data show thatthe panel of selected anti-DCIR mAbs had a range of DCIR bindingaffinities and properties.

The pure mAbs were also tested for FACS reactivity, firstly against 293cells transiently transfected with expression plasmid encodingfull-length DCIR, and then against various types of cultured and ex-vivohuman DC. The figure below shows a representative set of mAbs titratedin a FACS analysis versus DCIR 293 cell (control cells were negative).

FIG. 3 shows that CD34-derived human DC of both CD14+ and CD1a+ subtypesexpress cell surface DCIR. These two DC subtypes have profoundlydifferent roles in directing humoral versus cytolytic immuneresponses—thus the presence of DCIR on both subtypes suggests thatantigen targeted to human DC via DCIR should elicit both types ofimmunity—an important feature of vaccines directed against, e.g., viralinfections.

FIG. 4 shows that DCIR is also expressed on three human DC subtypesisolated directly from human skin. This observation shows that for DCIRantigen targeting vaccines, administration into the skin should beadvantageous since these DC types all express the receptor. It is knownthat these DC types are analogous to the above cultured human DC regardstheir immune directing properties and therefore targeting antigenthrough DCIR-bearing skin DC should be advantageous for elicitingdesirable mixed immune responses.

Dermal DCs and LCs were purified from normal human skin specimens.Specimens were incubated in the bacterial protease dispase type 2 for 18h at 4° C., and then for 2 h at 37° C. Epidermal and dermal sheets werethen separated, cut into small pieces (˜1-10 mm) and placed in RPMI 1640supplemented with 10% fetal bovine serum (FBS). After 2 days, the cellsthat migrated into the medium were collected and further enriched usinga Ficoll-diatrizoate gradient, 1.077 g/dl. DCs were purified by cellsorting after staining with anti-CD1a FITC and anti-CD14 APC mAbs.

Presence of DCIR in other human tissues. FIG. 5 shows DCIR-specificstaining of a population of cells surrounding a germinal center within ahuman tonsil. These cells are likely to be either resident DC or DCrecently migrated to this site after e.g., loading with foreign antigenand activation. The staining shows that administration of DCIR-targetedvaccines by routes other than skin permitting access to organs in whichimmunity is generated should also be advantageous for eliciting immuneresponse

Using anti-DCIR mAbs to target antigen to human DC. Flu M1 proteins werechemically cross-linked to mAbs using sulfosuccinimidyl6-[3′(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP; Pierce)according to the manufacturer's protocol. The multi-step protocolinvolved the activation of the mAb by modification of its amines throughthe NHS ester group of SPDP for 30 min at room temperature followed bydialysis versus PBS. Subsequently, Flu M1 proteins, which contain twofree sulfhydryl groups, were added and incubated at room temperatureovernight. The efficiency of the cross-linking reaction was estimated bycomparing the amount of Flu M1 protein before the reaction to the mAb,to the mAb/Flu M1 ratio after cross-linking. We calculated that, onaverage, 50% of the mAbs had reacted to one Flu M1 molecule. FIGS. 6 and7 show examples of the cross-Flu M1 protein and the mAb to DCIR.Analysis via reduced SDS-PAGE identified products with 1-2 Flu M1 permAb based on the ratio of staining of Flu M1/H chain and thesepreparations were used in the in vitro studies. Non-reduced SDS-PAGEanalysis (second figure below shows that the complexes were largelybetween Flu M1 and single mAbs as evidenced by a low percentage of verylarge complexes.

FIG. 6 shows the cross-linking of Coh.Flu M1 to Anti-DCIR_(—)2C9 mAb.Reduced SDS-PAGE analysis of cross-linked products purified by protein GSepharose affinity. From left to right are 2.5 μg, 1 μg Coh.Flu M1, 10μg products from reacting Coh.Flu M1 with mAb at ratios of 1:1, 2:1,4:1.

FIG. 7 shows the cross-linking of His.Flu M1 to mAbs. Non-reducedSDS-PAGE analysis of cross-linked products purified by protein GSepharose affinity. From left to right are 5 μg, His.Flu M1, followed bypairs of 5 μg mAb (anti-CD1a_OKT6, anti-LANG_(—)2G3, anti-DCIR_(—)2C9)and 5 μg mAb reacted with of 5 μg His.Flu M1.

Anti-DC receptor mAbs cross-linked to Flu M1 protein effectively targetthe antigen to human DC—Anti-DC receptor mAbs were chemicallycross-linked to Flu M1 protein and various doses were added to theco-culture of human CD34-derived CD1a+ DCs with autologous CD8+ T cells.CD40L was added to the culture after 24 h for DCs activation, followedby addition of IL-2, at day 3, for T cell proliferation. After 8-10days, T cells specific for the Flu M1 peptide GILGFVFTL (SEQ ID NO.: 53)were assessed by MHC tetramer analysis. FIG. 8 shows that Flu M1cross-linked to anti-DCIR mAb elicited the proliferation of FluM1-specific cells, while significantly less proliferation of FluM1-specific cells was observed with non-cross-linked Flu M1 and mAb atsimilar doses. The dose-ranging shows that the cross-linked mAb eliciteda response at least 50-fold more effectively than free Flu M1. This datademonstrates antigen-targeting, i.e., potentiation of an immuneresponse—in this case a recall of T cells with memory of a specific FluM1 epitope. CD34-DCs were sorted into CD1a+LCs or CD14+IntDCs subsets.FIG. 9 shows that anti-DCIR-targeted CD1a+LCs were much more potent atdirecting the expansion of Flu M1-specific CD8+ cells, despite similarlevels of DCIR expression on both cell types.

FIG. 8 shows that Flu M1 cross-linked to anti-DCIR mAb induces theexpansion of Flu M1-specific CD8+ T cells more efficiently than Flu M1protein unlinked to mAb. CD34-derived CD1a+ DCs were incubated with CD8+T cells and the indicated concentrations of anti-DCIR_(—)2C9 mAbcross-linked to His.Flu M1 or with unlinked mAb. CD8+ T cells were thenanalyzed for Flu M1-specific expansion. The inner boxes indicate thepercentages of tetramer-specific CD8+ T cells.

FIG. 9 shows that Flu M1 cross-linked to anti-DCIR mAb induces theexpansion of Flu M1-specific CD8+ T cells more efficiently via LCs thanInt-DCs. LCs or Int-DCs from an HLA-A2 donor and autologous CD8+ T cellswere co-cultured with the indicated concentrations of anti-DCIR_(—)2C9mAb cross-linked to His.Flu M1. Cross-presentation efficiency wasassessed by the frequency of Flu M1-specific CD8+ T cells and analyzedwith HLA-A201/pMI tetramer. The inner boxes indicate the percentages oftetramer-specific CD8+ T cells.

Development of recombinant anti-DCIR mAbs (rAbs) as prototypeantigen-targeting vaccines. Vectors were developed for the expression intransiently transfected mammalian cells of secreted anti-DC receptorrAbs that were chimeras of the mouse hybridoma-encoded H and L chainvariable (V) regions and human IgK or human IgG4H constant (C) regions.V regions from L and H chains of anti-DC receptor mAbs with differentspecificities (I.e., from different anti-DCIR hybridomas) were cDNAcloned, characterized by DNA sequence analysis, and engineered intothese vectors. FIG. 10 shows such H+L chain vectors encoding chimericmouse-human rAbs corresponding to a number of different anti-DCIR mAbsco-transfected into 293 cells and assayed by anti-human FC ELISA forsecretion of rAb into the culture supernatant.

The anti-DCIR rAbs encoded a ˜9.5 kDA dockerin domain in-frame with therAb H chain. The purpose of the dockerin domain (called rAb.Doc) is topermit assembly of specific [rAb.Doc:Coh.antigen] complexes. In thiscase, Coh.antigen refers to a fusion protein between a ˜17.5 kDa cohesindomain and an antigen. High affinity interaction between cohesin anddockerin is used to assemble defined complexes that we have showndeliver antigen to the surface of DC bearing the receptor specificity.For example, the figure below shows [anti-DCIR.Doc:Coh.Flu M1] complexesbound to the surface of human DC (here the Coh.Flu M1 is biotinylatedand detected on the cell surface after washing steps). ControlrAb.Doc:Coh.Flu M1 complexes (shown in red in the figure below) did notbind any more than the detecting streptavidin-PE reagent alone.

DCIR internalizes antigen with slow kinetics and this distinguishes itfrom other DC receptors. DC receptors such as DC-SIGN are characterizedby a rapid kinetics of internalization. For example, FIG. 11 shows thatanti-DC-SIGN/L.Doc internalizes Alexa-labeled Coh.Flu M1 into GM-CSF/IFNcultured human DC rapidly—most of the label is internal to the cellswithin 15 min. In contrast, anti-DCIR.Doc internalizes the Coh.Flu M1very slowly—at 3 hours there is both significant amounts of internalantigen and of cell-surface antigen. This result distinguishes DCIR as aslow-internalizing DC receptor and is in contrast to the conclusions ofBates et. al., who suggested that “Following cross-linking, DCIR wasonly slowly and weakly internalized in monocyte- and CD34-derived DC, incontrast to the rapid kinetics observed with the MMR (data not shown).This finding suggests that Ag capture by receptor-mediated endocytosisis not the principal function of DCIR”.

FIG. 11 shows that Coh.Flu M1 linked to anti-DCIR.Doc rAb bindsspecifically to GM/IL-15 human DC. Monocyte-derived GM-CSF/IL-15cultured human DCs were incubated with the indicated concentrations ofanti-DCIR.Doc rAbs premixed for 1 hour with a 4-fold molar excess ofbiotinylated Coh.Flu M1. After 1 hour, cells were washed and incubatedwith streptavidin-PE. After another wash, the cells were analyzed byFACS to detect cell-associated PE. Green plots are the Anti-DCIR.DocrAbs, red curves are control IgG4.Doc complexes.

FIG. 12 shows that the Coh.Flu M1 linked to anti-DC-SIGN/L.Doc oranti-DCIR.Doc rAb binds and is internalized into to GM-CSF/IL-4 humanDC. Monocyte-derived GM-CSF/IL-4 cultured human DCs were incubated withanti-DCIR.Doc or anti-DC-SIGN/L.Doc rAb premixed for 1 hour with a4-fold molar excess of Alexa-labeled Coh.Flu M1. After 1 hour on ice,cells were washed and placed at 37 C. Confocal microscopy was used toanalyze the cellular location of cell-associated antigen (shown in red).Green marks cell membrane-associated actin.

Targeting Coh.Flu M1 to human DC via DCIR.Doc identifies DCIR as asuperior receptor for vaccine development purposes. Targeting Flu M1antigen to human DC via the slow-internalizing DCIR receptor wascompared to targeting via fast-internalizing ASGPR and LOX-1 receptors.The immune response monitored was expansion of Flu M1-specific CD8+ Tcells. The results show targeting through DCIR is significantly moreefficacious than via LOX-1 or ASGPR. In a similar experiment, thesuperiority of targeting via DCIR was even more evident when the DC werewashed free of residual [rAb.Doc:Coh.antigen] before culture with theCD8+ T cells. This situation is likely closer to the in vivo situation,where targeted DC would move away from residual administered antigen toencounter T cells in draining lymph nodes.

FIG. 13 shows that the Anti-DCIR.Doc:Coh.Flu complex is more efficientat expanding Flu M1-specific CD8+ T cells than other [anti-DC receptorrAbs.Doc:Coh.Flu M1] complexes. CD34-derived CD1a+DCs, were co-culturedwith CD8+ T cells and 8 nM (top panel) or 0.8 nM (lower panel) of[anti-DCIR_(—)2C9.Doc:Coh.Flu M1], anti-LOX1_(—)15C4.Doc,anti-ASGPR_(—)49C11.Doc or IgG4.Doc control rAb, each complexed withCoh.Flu M1. CD8+ T cells were then analyzed for Flu M1-specificexpansion. The inner boxes indicate the percentages of tetramer-specificCD8+ T cells.

FIG. 14 shows that the Anti-DCIR.Doc:Coh.Flu complex administered for 1day is more efficient at expanding Flu M1-specific CD8+ T cells thanother [anti-DC receptor rAbs.Doc:Coh.Flu M1] complexes. Study conditionswere as for the figure above, expect DC were washed at day 1 prior toaddition of autologous CD8+ T cells. Some anti-DCIR V regions areparticularly favorable to secretion of important antigens fused at therAb H chain C-terminus.

FIG. 15 shows that various antigens expressed as fusions to theC-terminus of rAb H chain have intrinsic effects on the secretion ofrAb.antigen. Here identical antigen coding regions were engineered onchimeric hIgG4 rAbs with two different mouse V region specificities.These expression constructs were co-transfected with appropriate L chainmouse V-region—hIgk constructs into 293F cells and secretion of rAb wasappraised after three days. Some rAb antigens were well expressed,others (including Flu HA5-1) very poorly. It should be expected thateach antigen has intrinsic biochemical properties affecting secretion inthe context of rAb. Indeed there is a strikingly parallel effect onexpression in the context of the two V region specificities tested.

Flu HA5 is an antigen that is important to consider in development of avaccine against avian influenza. FIG. 16 shows the unexpected discoverythat different anti-DCIR V regions (derived from different anti-DCIRmAbs) greatly affect secretion of the desired anti-DCIR.Flu HA5 vaccine.In the example shown below, DCIR_(—)25A4 is particularly favorable forsecretion of this type of vaccine when compared to other DCIR V regions.

FIG. 16 shows the Anti-DCIR.Flu HA5 rAbs are secreted at variousefficiencies depending on the nature of the variable regions. H and Lchain expression plasmids encoding chimeric mouse V region and human Cregion fused via the H chain C-terminus to either Doc (Blue circles) orHA5-1 (Red triangles) were co-transfected into 293 cells and after 3days dilutions of the supernatants were assayed for IgGFc by ELISA.Except for DCIR_(—)2C9, rAb.Docs were generally well expressed. However,expression of rAb.HA5-1s varied widely.

The unique property of the anti-DCIR 25A4 V-regions to favor secretionof rAb.HA5-1 illustrates application of claim 5. That is based on ourinvention that a particular V-region can affect secretion of arAb.antigen. This means that intrinsic poor secretion of a particularantigen in the context of a rAb fusion protein can be overcome byscreening different V regions with the desired combining specificitiesfor those favorable for secretion. This is claimed as a new generalprinciple for any secreted rAb.fusion protein.

Anti-DCIR enhances priming of HIV specific CD8+ T cells. FIG. 17 showsthat anti-DCIR mAb has a particular action on dendritic cells thatenhances priming—that is the uptake of peptide and its presentation onsurface MHC to T cells specific to the peptide antigen. The exampleshows that stimulation of DC with anti-DCIR mAb together with CD40L, aknown DC activation signal usually delivered by cognate T cells,resulted in greatly increased numbers of CD8+ T cells specific to theimmunodominant HIV gag peptide that was added to the DC culture. Thisproperty is highly predictive for successful antigen targeting viaanti-DC receptor rAb vaccines and indicates that anti-DCIR.antigenvaccines will be superior.

FIG. 17 shows that Anti-DCIR mAb enhances priming of HIVantigen-specific CD8⁺ cells. Purified total CD8⁺ T cells (2×10⁶cells/well) were stimulated with autologous IFN-DCs (1×10⁵ cells/well)and HLA-A201-restricted HIV peptides pol (pol₄₇₆₋₄₈₄ ILKEPVHGV (SEQ IDNO.: 54), pol₂₉₃₋₃₀₂ KYTAFTIPSI (SEQ ID NO.: 55)), and gag (gag₇₇₋₈₅,SLYNTVATL (SEQ ID NO.: 56), gag₁₅₁₋₁₅₉, TLNAWVKVV(SEQ ID NO.: 57)) (5μM). Cells were cultured for 9 days in 24 well plates that werepre-coated over night at 4 c with 5 ug/well Anti-DCIR mAbs or controlmonoclonal antibodies diluted in PBS pH 9.6 and washed extensively.Cells were cultured in Yssel's medium supplemented with 10% human ABserum, 10 U/ml IL-7 (R&D) and 100 ng/ml CD40L (R&D). IL-2 was added at10 U/ml at day 3. Expansion of peptide-specific CD8⁺ T cells wasdetermined by counting the number of cells binding peptide/HLA-A201tetramers (Beckman Coulter) at the end of the culture period

Anti-DCIR mAb enhances cross priming. FIG. 18 shows that anti-DCIR mAbhas a particular action on dendritic cells that enhancescross-priming—that is the uptake of protein and its correct processingand presentation on surface MHC as measured by expansion of T cellsspecific to the antigen-derived peptide. The example shows thatstimulation of DC with anti-DCIR mAb together with CD40L, a known DCactivation signal usually delivered by cognate T cells, resulted ingreatly increased numbers of CD8+ T cells specific to the immunodominantMART-1 epitope from the Cohesin-MART-1 fusion protein that was added tothe DC culture. This property is highly desirable for antigen targetingvia anti-DC receptor rAb vaccines and indicates that anti-DCIR.antigenvaccines will be superior.

FIG. 18 shows the Anti-DCIR mAb enhances priming of HIV antigen-specificCD8+ cells. Above Figure method as for the previous figure, except thatcoh.MART-1peptide fusion protein replaced peptide.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

FIG. 19 shows an immunohistochemistry analysis of DCIR distribution inhuman epithelial sheet. DR-FITC staining is shown in green and PAB269(DCIR)-568 is shown in red. The upper right panel shows the imagessuperimposed. Blue staining is DAPI for cell nuclei. Digital imaging@40×. The cell morphology and DR staining is characteristic of epidermalLangerhans cells—thus, the analysis reveals DCIR expression onLangerhans cells—pointing to the utility of DCIR to uptakeanti-DCIR.antigen conjugate applied to e.g. scarified skin. Therefore,these data shows that Langerhans cells uptake of antigen associated withDC activation via adjuvant will result in potent cellular responsesagainst the targeted antigen.

FIGS. 20A-20D shows monoclonal antibodies to DCIR Antigen, specifically,affinity to DCIR.

Immobilization of DCIR antigen: DCIR antigen was immobilized onto AKT_ivcovalent sensor surfaces via the primary amines (50 ug·mL-1 in 10 mMsodium acetate, pH 5.5). The carboxylate surfaces were activated using amixture of EDC and NHS and DCIR was coupled to all four channels.Finally, any remaining carboxylate groups were deactivated using aproprietary blocking agent.

Determination of affinities of four anti-DCIR antibodies in HBS: Todetermine the affinities of four anti-DCIR antibodies, dilution seriesof each antibody were prepared from 10 to 0.3125 μg ml-1 and injected inparallel over immobilized DCIR antigen for 180 s. Between sampleinjections the surfaces were regenerated using two 60 s injections of100 mM hydrochloric acid. Biosensor measurements were performed on anAkubio acoustic biosensor.

TABLE 1 Kinetic Parameters calculated from the interaction of fourmonoclonal antibodies with immobilized DCIR antigen. k_(a) Antibody(M⁻¹s⁻¹ × 10⁵ k_(d) (s⁻¹ × 10⁻⁴) K_(D) (pM) Hybridoma Anti- 2.07 1.15560 DCIR24A5.4A5 rAb Anti- 2.38 3.26 1370 DCIR24A5.4A5.DocVar1 C377Hybridoma Anti- 5.56 4.70 850 DCIR29E9.2E2 rAb Anti- 1.50 2.90 1940DCIR29E9.2E2.DocVar1 C409

Table 1 shows the high affinity DCIR ectodomain binding properties oftwo preferred anti-DCIR monoclonal antibodies 24A5 and 9E8 anddemonstrate that the derived mouse variable regions, when grafted onto ahuman IgG4 body, largely retain the high affinity binding properties.This data supports the specific claim to the sequences [and their‘humanized’ derivatives] of these variable regions regards utility forbinding to human DCIR.

Cross-reactivity of anti-DCIR mAbs to Rhesus macaque DCIR. To test thecross-reactivity of anti-human DCIR mAbs to Rhesus macaque DCIR, 233Fcells were transfected with Rhesus macaque DCIR cDNA configured into amammalian expression vector. The panel of anti-human DCIR antibodies wastested via FACS for binding to monkey DCIR compared to untransfected293F cells and 293F cells transfected with an identical vector directingthe expression of human DCIR. A comparison between the human and monkeyDCIR sequences is shown below. Those antibodies showing cross-reactivitybetween human and monkey DCIR are particularly preferred since, whenconfigured e.g., as recombinant humanized anti-DCIR.antigen vaccine, asa therapeutic agent, NHP toxicity studies can also addressmechanism-based issues [i.e., these testes can also address efficacyrelative to toxicity].

Human vs. Monkey DCIR. The primary sequence show is human and changesseen in monkey DCIR are shown below the human sequence. The putativetransmembrane region is highlighted in underlined. Non-conservativechanges are shown highlighted in bold.

MTSEITYAEVRFKNEFKSSGINTASSAASKERTAPHKSNTGFPKLLCASLLIFFLLLAISFFIAFVIFFQKYSQLLEKKT(SEQ ID NO.: 58) MTSEITYAEVRQNESKSSGIDSASSAASKKRTAPHKSNTGFSKLLCASLMIFFLLLAISFFFAFFIFFQKYSQLLEKMT(SEQ ID NO.: 59)TKELVHTTLECVKKNMPVEETAWSCCPKNWKSFSSNCYFISTESASWQDSEKDCARMEAHLLVINTQEEQDFIFQNLQEE(SEQ ID NO.: 60)TKDLVHTTLECVKKNMTTEETAWSCCPKNWKPFSSNCYFISTESASWQKSEKDCARMEAHLLVINTREEQDFIFQNLQEE(SEQ ID NO.: 61)SAYFVGLSDPEGQRHWQWVDQTPYNESSTFWHPREPSDPNERCVVLNFRKSPKRWGWNDVNCLGPQRSVCEMMKIHL(SEQ ID NO.: 62)SAYFVGLSDPEGQRHWQWVDQTPYNESSTFWHPHEPSDPDERCVVLNFRKTPKRWGWNDVHCIVPQRSVCEMMKIHL(SEQ ID NO.: 63)

FIG. 21 shows the cross-reactivity of anti-DCIR mAbs to Rhesus macaqueDCIR. A sample FACS analysis is presented below. Green plots show thebackground binding by control IgG4.gag recombinant protein. The red pltsare binding via anti-DCIR.gag proteins [secondary antibody wasPE-labeled antihuman IgGFc]. The result shows comparable binding by 9E8and 24A5 mAbs on 293F cells transfected with human DCIR expressionplasmid—on 293F cells transfected with monkey DCIR expression plasmid,9E8, but not 24A5, bound. In a similar analysis mAbs 9E8, 29G10, 31A6,3C2 bound well to monkey DCIR, but mAbs 24A5, 6C8, 24E7, 5F9, 29E9 didnot bind.

FIG. 22 is a graph that shows the binding of DCIR ectodomain to specificglycan structures. DCIR ectodomain was expressed as a hIgGFc fusionprotein secreted from 293F cells and was purified by protein A affinitychromatography. The protein was tested for binding of specific glycansusing the version 3.0 of the printed array from the Consortium forFunctional Glycomics—this array consists of 320 glycans (or glycoforms)in replicates of 6. The Excel spread sheet shown below presents incolumns A-F, respectively, the Glycan number, the structure or name, theaverage RFU value from the 6 replicates, the standard deviation, thestandard error of the mean (used for the error bars in the graph above,which presents the entired data set) and % CV. Columns C-Y contain thegraph of glycan number vs. Average RFU, and Columns Z-AE is the datafrom A-F sorted by RFU (high to low) to provide a list of the Glycansbound with highest intensity. The highest and lowest point from each setof six replicates has been removed so the average is of 4 values ratherthan 6. This eliminates some of the false hits that contain a singlevery high point. Thus, points with high % CV should be consideredsuspect. The analyses was done with detection using anti-human IgG-Fcthat was labeled with Phycoerythrin. The DCIR.IgFc was diluted in PBS to200 μg/ml using Tris-saline binding buffer containing 2 mM Ca and Mg, 1%BSA and 0.05% Tween 20.

This data was generated in collaboration with the Functional GlycomicsConsortium.Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12was the glycan which bound DCIR ectodomain most tightly. Other hIgGFcfusion proteins tested did not show a preference for this glycan whichis a very complex carbohydrate found on several human serum proteins.

Thus, antigen decorated with glycan 143, or a higher affinity derivativescreened from a panel of related structures, should hone the antigen toDCIR and serve as a surrogate for the anti-DCIR component of theDC-targeting vaccine or other DCIR targeting agent. This could have costbenefit in vaccine manufacture and storage.

TABLE 2 DCIR.IgGFc @ 200 ug/ml vs. anti-Human IgG PE StDev SEM Avg w/ow/o w/o Glycan Max & Max & Max & number Glycan name Min Min Min % CV 143Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3-4GlcNAc 38314 2007 1004 52Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12 173GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ-Sp8 17109 1865 932 11 172 (GlcNAcβ1-4)5β-Sp811161 1247 624 11 45 [6OSO3]Galβ1-4[6OSO3]Glcβ-Sp8 9762 1375 688 14 42[6OSO3]Galβ1-4Glcβ-Sp0 8376 3206 1603 38 15 α-Neu5Ac-Sp11 8128 887 44311 92 GalNAcβ1-4GlcNAcβ-Sp0 8066 1278 639 16 47 [6OSO3]GlcNAcβ-Sp8 79062365 1183 30 1 Neu5Acα2-8Neu5Acα-Sp8 7738 1337 669 17 29[3OSO3]Galβ1-4(6OSO3)Glcβ-Sp0 7572 993 496 13 30[3OSO3]Galβ1-4(6OSO3)Glcβ-Sp8 7515 538 269 7 271Fucα1-2[6OSO3]Galβ1-4[6OSO3]Glc-Sp0 7400 794 397 11 216Neu5Acα2-3Galβ1-3(6OSO3)GlcNAc-Sp8 7334 3242 1621 44 26[3OSO3][6OSO3]Galβ1-4[6OSO3]GlcNAcβ-Sp0 7193 939 470 13 171(GlcNAcβ1-4)6β-Sp8 7154 1358 679 19 286 [3OSO3]Galβ1-4[6OSO3]GlcNAcβ-Sp07146 1267 633 18 265 [3OSO3]Galβ1-4(Fucα1-3)(6OSO3)Glc-Sp0 7143 571 2858 *** Glycan #143 isNeu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12.

FIG. 23A to 23C show that DCIR is a global target for all blood DCsubsets. Two subsets of DCs are identified in the blood: CD11c⁺mDCs andBDCA2⁺pDCs. DCIR is one of the rare lectin-type receptors found on bothDC subsets. mDCs and pDCs were purified from cytapheresis and each DCsubset was cultured with autologous purified CD8⁺T cells and decreasingconcentrations of four recombinant forms of Flu-MP: Flu-MP, Flu-MP fusedto IgG4 and Flu-MP fused to two different recombinant anti-DCIRantibodies: 24A5 and 9E8.

Results shown in FIG. 23A below indicate that both recombinantDCIR-Flu-MP fusion proteins can potently target Flu-MP to mDC as the twoproteins can induce between 1.78% and 2.18% tetramer positive cells at aconcentration as low as 80 pM, a point where Flu-MP itself and theIgG4-Flu-MP are not able to induce expansion of antigen-specific Tcells. pDCs were also able to crosspresent the four forms of recombinantFlu-MP at 8 nM. At 0.8 nM and 80 pM, two DCIR-Flu-MP constructs werecrosspresented but the two other Flu-MP constructs were not (Fig. Bbelow).

Taken together, these data indicate that DCIR potently target proteinsfor crosspresentation by both blood mDCs and pDC. In human LCs andIntDCs have capacity to preferentially, respectively, prime cellularimmunity and humoral immunity. Targeting antigen to a pan-DC molecule,like DCIR, will potentially induce a wide humoral and cellular immuneresponse by targeting various DC subsets. This is in contrast to asubset-specific antigen-delivery vehicle such as anti-Langerin.

FIG. 23A shows blood-derived mDCs from HLA-A2 donor are targeted with 8nM, 0.8 nM or 80 pM each of aDCIR-Flu-MP (a#24A5 and b#9E8),IgG4-Flu-MP, or Flu-MP, matured with CD40L and co-cultured withautologous CD8⁺ T cells. 10 d later, T cell expansion evaluated byspecific HLA-A2-M1 tetramer staining [vertical axis].

FIG. 23B shows blood-derived pDCs from HLA-A2 donor are targeted with 8nM, 0.8 nM or 80 pM each of aDCIR-Flu-MP (a#24A5 and b#9E8),IgG4:Flu-MP, or Flu-MP, matured with CD40L and co-cultured withautologous CD8⁺ T cells. 10 d later, T cell expansion evaluated byspecific HLA-A2-M1 tetramer staining [vertical axis].

FIG. 23C shows DCIR allows crosspresentation of proteins by LCs anddermal CD14⁺DCs. Skin-derived DC from HLA-A2 donor are targeted with 8nM each of anti-DCIR:Flu-MP, anti-Langerin:Flu-MP or IgG4:Flu-MP,matured with CD40L and co-cultured with autologous CD8⁺ T cells. 10 dlater, T cell expansion was evaluated by specific HLA-A2-M1 tetramerstaining [vertical axis].

FIG. 24 shows that demonstrate that vaccination with DCIR-FluM1 permitsgeneration of FluM1 specific recall CD8+ T cell immunity. The resultsfrom sublethally irradiated NOD/SCID β2m−/− immunodeficient mice weretransplanted with 3×10⁶ CD34+ HPCs from HLA-A*0201+ healthy donors, andat 4-8 weeks post transplantation reconstituted by adoptive transfer of20×106 autologous T cells. Mice were pre-treated for 10 days with fivedoses of human recombinant FLT3-ligand (FLT3-L) to mobilize DCs. A totalof 30 mcg DCIR-FluM1 vaccine was delivered in two sites: i.p. and i.v.at two time points, i.e, day 1 and day 7 with 50 mcg/mouse poly IC asadjuvant. Induction of influenza-specific immune response was assessedby staining blood and tissues with matrix protein 1: FluM158-66(GILGFVFTL) (SEQ ID NO.: 64) peptide-loaded tetramer. As shown in FIG.1, 4/5 mice vaccinated with DCIR-FluM1 demonstrated, at day 11 postvaccination, circulating human CD8+ T cells binding FluM1 tetramer:0.63%, 0.34%, 0.21%, and 0.62%. Staining with control tetramer loadedwith HIV gag peptide was nearly negative. These preliminary results wereconfirmed in independent cohorts of mice and the expansion of highaffinity FluM1 tetramer-binding CD8+ T cells was observed in a total of9/12 vaccinated mice. These results demonstrate that vaccination withDCIR-FluM1 permits generation of FluM1 specific recall CD8+ T cellimmunity.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1. A method for increasing the effectiveness of antigen presentation bya DCIR-expressing antigen presenting cell comprising the step ofisolating and purifying a DCIR-specific antibody or fragment thereof towhich a targeted agent is attached that forms an antibody-agent complex,wherein the molecule is internalized by the antigen presenting cellsthat has been contacted with the antibody-agent complex.
 2. The methodof claim 1, wherein antigen presenting cell comprises a dendritic cell.3. The method of claim 1, wherein DCIR-specific antibody or fragmentthereof is bound to one half of a Coherin/Dockerin pair.
 4. The methodof claim 1, wherein DCIR-specific antibody or fragment thereof is boundto one half of a Coherin/Dockerin pair and a targeted agent is bound tothe complementary half of the Coherin/Dockerin pair to form a complex.5. The method of claim 1, wherein the targeted agent is selected from apeptide, protein, lipid, carbohydrate, nucleic acid, and combinationsthereof.
 6. The method of claim 1, wherein the targeted agent comprisesone or more cytokines.
 7. The method of claim 6, wherein the targetedagent comprises one or more cytokines selected from interleukins,transforming growth factors (TGFs), fibroblast growth factors (FGFs),platelet derived growth factors (PDGFs), epidermal growth factors(EGFs), connective tissue activated peptides (CTAPs), osteogenicfactors, and biologically active analogs, fragments, and derivatives ofsuch growth factors, B/T-cell differentiation factors, B/T-cell growthfactors, mitogenic cytokines, chemotactic cytokines, colony stimulatingfactors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4,IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17,IL18, etc., leptin, myostatin, macrophage stimulating protein,platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL,human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10,PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF,transforming growth factor (TGF) supergene family include the betatransforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bonemorphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblastgrowth factor (FGF), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (forexample, Inhibin A, Inhibin B); growth differentiating factors (forexample, GDF-1); and Activins (for example, Activin A, Activin B,Activin AB).
 8. The method of claim 1, wherein the targeted agentcomprises a bacterial, viral, fungal, protozoan or cancer protein.
 9. Amethod for increasing the effectiveness of antigen presentation bydendritic cells comprising binding a DCIR-specific antibody or fragmentthereof to which an antigen is attached that forms an antibody-antigencomplex, wherein the antigen is processed and presented by a dendriticcell that has been contacted with the antibody-antigen complex. 10.(canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled)
 14. A method forincreasing the effectiveness of dendritic cells comprising: isolatingpatient dendritic cells exposing the dendritic cells to activatingamounts of anti-DCIR antibodies or fragments thereof and antigen to formantigen-loaded, activated dendritic cells; and reintroducing theantigen-loaded, activated dendritic cells into the patient.
 15. Themethod of claim 14, wherein the antigen comprises a bacterial, viral,fungal, protozoan or cancer protein.
 16. An anti-DCIR immunoglobulin orportion thereof that is secreted from mammalian cells and an antigenbound to the immunoglobulin.
 17. The immunoglobulin of claim 16, whereinthe immunoglobulin is bound to one half of a cohesin/dockerin domain.18. The immunoglobulin of claim 16, further comprising a complementaryhalf of the cohesin-dockerin binding pair bound to an antigen that formsa complex with the modular rAb carrier.
 19. The immunoglobulin of claim16, further comprising a complementary half of the cohesin-dockerinbinding pair that is a fusion protein with an antigen.
 20. Theimmunoglobulin of claim 16, wherein the antigen specific domaincomprises a full length antibody, an antibody variable region domain, anFab fragment, a Fab′ fragment, an F(ab)₂ fragment, and Fv fragment, andFabc fragment and/or a Fab fragment with portions of the Fc domain. 21.The immunoglobulin of claim 16, wherein the immunoglobulin is bound to atoxin selected from wherein the toxin is selected from the groupconsisting of a radioactive isotope, metal, enzyme, botulin, tetanus,ricin, cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins,shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin,abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venomand spider venom.
 22. The immunoglobulin of claim 16, wherein theantigen is a fusion protein with the immunoglobulin.
 23. A vaccinecomprising a DCIR-specific antibody or fragment thereof to which anantigen is attached that forms an antibody-antigen complex, wherein theantigen is processed and presented by a dendritic cell that has beencontacted with the antibody-antigen complex.
 24. The vaccine of claim23, wherein the antigen comprises DCIR.
 25. A T cell antigen comprising:an antigenic T cell epitope peptide bound to at least a portion of aglycan comprisingNeu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-3(Neu5Acα2-3Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ-Sp12that binds specifically to DCIR.